Abstract: Objective To explore the effect and mechanism of hyperoside on high glucose-induced oxidative stress injury of myocardial cells. Methods Oxidative stress injury of myocardial cells was simulated by treating with high glucose. Cells were divided into five groups:control group (5.5 mmol/L glucose); high glucose model group (35 mmol/L glucose); hyperoside protection groups (35 mmol/L glucose +4, 8, 20 nmol/L hyperoside). Cells were incubated for 48 h. The cell viability was detected by CCK-8. Apoptosis was measured through flow cytometry. The level of ROS was tested by Reactive Oxygen Species Assay Kit DCFH-DA with flow cytometer. The level of SOD and MDA was detected by SOD Assay Kit and MDA Assay Kit respectively. The protein levels of phosphatidylinositide 3-kinases (PI3K), protein kinase B (AKT), p-AKT, nuclear factor erythroid 2-related factor 2 (Nrf2) and p-Nrf2 were detected by Western blot. The activation of AKT was analyzed by immunofluorescence staining. Results Compared with control group, the cell viability, the levels of SOD, the expression of PI3K, the ratio of p-AKT/AKT and p-Nrf2/Nrf2 and the percentage of AKT positive cells in high glucose group were decreased with enhancive apoptosis and levels of ROS and MDA (P<0.05). Compared with high glucose group, the cell viability, the levels of SOD, the expression of PI3K, the ratio of p-AKT/AKT and p-Nrf2/Nrf2 and the percentage of AKT positive cells in hyperoside group (4, 8, 20 nmol/L) were increased with reduced apoptosis and levels of ROS and MDA (P<0.05). Conclusion Hyperoside protects myocardial cells against oxidative stress injury via activation of PI3K/AKT/Nrf2 signal pathway.